CN111269296B - nLsA protein, structural gene thereof and application thereof - Google Patents
nLsA protein, structural gene thereof and application thereof Download PDFInfo
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- CN111269296B CN111269296B CN202010151094.8A CN202010151094A CN111269296B CN 111269296 B CN111269296 B CN 111269296B CN 202010151094 A CN202010151094 A CN 202010151094A CN 111269296 B CN111269296 B CN 111269296B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
- A01N47/44—Guanidine; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
Abstract
The invention relates to the field of genetic engineering, and particularly provides an nLsA protein, a structural gene thereof and application thereof. The nLsA protein is a1) or a 2): a1) the amino acid sequence is protein shown as SEQ ID No. 2; a2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in a1) and has the same functions as the a1) sequence. Compared with the commonly-used commercialized nisin, the nLsA protein has better bactericidal effect, can obtain better use effect under the condition of the same use amount, and has huge potential application value.
Description
Technical Field
The invention relates to the field of genetic engineering, and in particular relates to an nLsA protein, a structural gene thereof and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Lactein is a bacteriostatic substance produced by lactic acid bacteria, and its structure is generally short-chain peptide. Common lactein such as Nisin has a broad antibacterial spectrum and can inhibit gram-positive bacteria. When the compound is used together with citric acid, polylysine, glycine and other substances, gram-negative bacteria can be effectively inhibited. The world health organization approved Nisin for use in 1969, approved by the U.S. FDA as a food preservative in 1988, and recognized Nisin as a generally recognized as safe (gras) substance. The inventor finds that compared with the artificially synthesized chemical preservative, the lactein is safer, can be quickly decomposed by trypsin in the digestive tract and does not influence the colony environment in the digestive tract. And, lactein also has potential in the medical field because it does not produce cross-resistance with medical antibiotics, can help to cope with some complex infectious diseases, enhance the healing effect; the application potential of the lactein is very large, if a novel lactein with high titer can be discovered, a better use effect can be obtained under the condition of the same use amount, and the lactein has great potential application value.
Disclosure of Invention
Therefore, the invention aims to provide an nLsA protein, a structural gene and an application thereof, wherein the nLsA protein is a novel lactein, the nlsA protein is coded by the nLsA gene and has excellent effects of killing and/or inhibiting bacteria, and the effect of the nlsA protein under the same action concentration is obviously superior to that of the commonly commercialized lactein at present.
Specifically, the technical scheme of the invention is as follows:
in a first aspect of the invention, the invention provides an nLsA protein that is a1) or a 2):
a1) the amino acid sequence is protein shown as SEQ ID No. 2;
a2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in a1) and has the same functions as the a1) sequence.
The nLsA protein has a molecular weight of 5-10kDa, preferably about 7 kDa.
In a second aspect of the invention, the invention provides a gene encoding said nLsA protein.
Wherein the gene has the nucleotide sequence of b1), b2) or b 3):
b1) a nucleotide sequence shown as SEQ ID NO. 1;
b2) a nucleotide sequence complementary to b 1);
b3) a nucleotide sequence which has more than or equal to 90 percent of identity with the nucleotide sequence shown in b1) or b2) and encodes the same functional protein.
Wherein the nucleotide sequence has a sequence identity of 90% or more and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (complete).
In a third aspect of the invention, the invention provides a primer pair for amplifying the gene encoding the nLsA protein (i.e. the gene as described in the second aspect above), designated nLsA-F and nLsA-R, wherein the nucleotide sequence of nLsA-F is shown as SEQ ID No.3 and the nucleotide sequence of nLsA-R is shown as SEQ ID No. 4.
In a fourth aspect of the invention, the invention provides a biological material comprising the gene encoding an nLsA protein (i.e. the gene as described in the second aspect above), which biological material is an expression cassette, a recombinant vector, a recombinant microorganism or a recombinant cell.
In a fifth aspect of the invention, the invention provides a method of constructing a recombinant strain containing the gene encoding nLsA protein (i.e. the gene as described in the second aspect above), comprising the steps of:
vector construction: taking nLsA-F and nLsA-R as primer pairs (namely the primer pairs in the third aspect) and taking the genome of the pickle bacteria as a template, amplifying to obtain nLsA genes, and performing double enzyme digestion and recombination connection to obtain nLsA expression vectors;
and (3) vector transformation: transferring the nLsA expression vector into competent cells and culturing the nLsA expression vector on a selective medium containing chloramphenicol;
screening positive clones: and selecting transformants with correct sequencing, and verifying by sequencing with upstream and downstream primers of the gene, wherein the recombinant strain obtained with correct sequencing is obtained.
In some embodiments of the invention, the method comprises the steps of:
designing primers nLsA-F and nLsA-R, wherein the primer sequences are respectively shown as SEQ ID No.3 and SEQ ID No. 4;
amplifying to obtain a fragment containing a gene sequence SEQ ID No.1 by taking the genome of the pickled vegetable bacteria as a template;
carrying out double enzyme digestion on the amplified fragment and a vector (such as pNZ8148 plasmid), and connecting the amplified fragment and the vector to the downstream of a constitutive promoter to construct an nLsA expression vector;
preparing lactic acid streptococcal competent cell, taking 50uL competent cell, adding 1ug of the above-mentioned constructed plasmid, standing on ice for a period of time to make it uniformly mixed, then making electric shock according to the electric conversion condition. Adding a recovery culture medium for culture after electric shock, centrifuging to remove supernatant after full recovery culture, adjusting to 10% of the original volume, then resuspending and coating on a selection culture medium containing 20ug/mL chloramphenicol resistance for culture; the selection medium can be GM17 medium, for example, the GM17 medium is obtained by adding 10g/L sterilized glucose solution to M17 medium, wherein the M17 medium is a commercial product and has the components of 5.0g/L of soybean peptone, 2.5g/L of casein peptone, 2.5g/L of yeast extract, 5.0g/L of beef extract, 5.0g/L of lactose, 0.5g/L of sodium ascorbate, 19.0g/L of sodium beta-glycerophosphate, and 0.25g/L, pH magnesium sulfate, and the value of 7.2 +/-0.2.
And (3) carrying out inverted culture at 30 ℃, screening positive transformants after 48-96 h, and further verifying the obtained positive transformants by using gene upstream and downstream primer sequencing.
In a sixth aspect, the present invention provides the use of the nLsA protein of the first aspect, the gene of the second aspect, or the biomaterial of the fourth aspect for the preparation of a product with bactericidal and/or bacteriostatic effects.
Wherein, the sterilization and/or bacteriostasis is to kill and/or inhibit gram-positive bacteria or gram-negative bacteria, preferably micrococcus luteus.
The product can be a bactericide, a bacteriostatic agent, a disinfectant, a hand sanitizer, a preservative, and the like, or can be a tangible object, such as a textile, a substrate, and the like, containing (e.g., added with, coated with, and the like) the nLsA protein of the present invention.
In a seventh aspect of the invention, there is provided a method of killing and/or inhibiting bacteria, the method comprising contacting the bacteria with an effective amount of the nLsA protein or the article of manufacture referred to in the sixth aspect above for a time sufficient to be effective to kill or inhibit the bacteria.
Wherein the bacteria are gram-positive bacteria or gram-negative bacteria, preferably micrococcus, especially micrococcus luteus.
Wherein the effective amount is not less than 0.1 mg/mL.
Compared with common commercial nisin, such as NisinA (coded by nisA gene) and NisinZ (coded by nisZ gene), the nLsA protein has the advantages of better sterilization and/or bacteriostasis effects, better use effect under the condition of equal use amount and huge potential application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a Tris-Tricine-SDS-PAGE of the nlsA protein (i.e. lactein) which is the expression product of the structural gene nlsA of lactein according to example 2 of the present invention.
FIG. 2 is a graph comparing the bacteriostatic effect of nLsA expression product with nisin NisinA and NisinZ.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The experimental procedures in the following detailed description, if specific conditions are not indicated, are generally performed according to conventional methods and conditions of molecular biology within the skill of the art, which are fully explained in the literature, see, for example, Sambrook et al, molecular cloning: techniques and conditions as described in the Experimental handbook or the molecular cloning guidelines, or as recommended by the manufacturer.
The preferred embodiments and materials described herein are intended to be exemplary only.
Example 1Amplification of the Lactein structural Gene nLsA
The method comprises the steps of carrying out amplification culture on the pickled vegetable bacteria obtained from a pickled vegetable jar, taking 5mL of bacterial liquid, carrying out lysozyme treatment, carrying out genome extraction by using a bacterial genome extraction kit, and obtaining the genome of the pickled vegetable bacteria by referring to a kit specification in the extraction step.
A pair of primers was designed by inserting restriction sites XbaI and HindIII (underlined) into the 5' end of the nLsA gene sequence shown in SEQ ID No.1, respectively. The primer sequences are as follows:
nLsA-F：5’-TGCTCTAGAATGAGTACAAAAGATTTTAAC-3’；
nLsA-R：5’-CCCAAGCTTTTATTTGCTTACGTGAATAC-3’；
the target gene is amplified by using the genome as a template and the sequence as a primer. The amplification process is carried out according to high-fidelity enzyme instructions, and the product is purified and recovered after the amplification is finished. The recovered product was digested with XbaI and HindIII (NEB, USA) and ligated with the plasmid pNZ8148 digested with XbaI and HindIII in a recombinant manner, and the recombinant reaction was carried out according to the product instructions. The recombinant plasmid was transformed into E.coli MC1061 competent cells, and positive clones were verified by sequencing on GM17 selection medium containing 20ug/mL chloramphenicol overnight.
And selecting transformants with correct sequencing, and extracting the plasmid pNZ8148-nLsA after amplification culture. Mixing the plasmid and competence, and transferring the plasmid into NZ9000 lactobacillus by electric conversion method. Culturing at 30 ℃, selecting a single clone for identification, and obtaining the obtained recombinant strain NZ9000-nLsA after the sequencing is correct.
Example 2Expression purification of Lactobacilli Gene
Well-grown recombinant bacteria (NZ9000-nLsA, example 1) were inoculated into 50mL of GM17 medium containing chloramphenicol, and after overnight culture, the cells were inoculated into 200mL of GM17 medium (containing chloramphenicol) at 10%, and inducible expression was performed by adding an inducible amount of Nisin. After fermentation for a certain period of time, the culture was centrifuged, and the supernatant was filtered through a 0.22um filter and then subjected to separation and purification of the lactein protein using an SP ion exchange column (GE, USA). The buffer solutions used for purification were as follows:
and (3) an equilibrium buffer: 50mmol/L lactic acid, pH adjusted to 3.0 with NaOH;
elution buffer: 50mmol/L of lactic acid, 400mmol/L of NaCl and 3.0 of NaOH adjusted pH;
the concentration of the collected protein after purification was tested by the Bradford method. And concentrating the purified protein solution, detecting by using Tris-Tricine-SDS-PAGE protein electrophoresis, and comparing with NZ9700 which is not transformed, so as to determine the expression condition of the nLsA protein.
The concentration of the electrophoresis separation gel is 16.5%, the concentration of the sandwich gel is 10%, the concentration of the concentrated gel is 4%, the electrophoresis result shows that the nLsA protein is successfully expressed, the protein molecular weight is about 7kDa, and the electrophoresis chart is shown in figure 1.
The GM17 medium was prepared by adding 10g/L sterilized glucose solution to M17 medium, wherein the M17 medium is a commercial product and comprises 5.0g/L of soybean peptone, 2.5g/L of casein peptone, 2.5g/L of yeast extract, 5.0g/L of beef extract, 5.0g/L of lactose, 0.5g/L of sodium ascorbate, 19.0g/L of sodium-beta-glycerophosphate, and 0.25g/L, pH magnesium sulfate, and has a value of 7.2. + -. 0.2.
Example 3Experiment of bacteriostatic effect of Lactobacilli
And (4) recovering the test bacteria, respectively inoculating the test bacteria (micrococcus luteus) into 10mL of LB culture medium, and performing shaking culture at a proper temperature overnight. Heating the corresponding solid culture medium, adding 1% of test bacterium liquid when the solid culture medium is cooled to about 45 ℃, uniformly mixing, pouring the mixture into a flat plate, wherein each plate contains about 20g of the culture medium, and drying the plate in a super clean bench. A total of 3 test plates were set up to test the NisinA, NisinZ, nLsA proteins separately, and a sterile punch was used to punch holes evenly through the test plates, 4 wells per plate, of which 1 was blank, and 3 replicates of the same sample (NisinA, NisinZ or nLsA protein) were tested in 3 wells, with 30uL of sample added per well. The flat plate is placed in an incubator at 37 ℃, cultured for 48h, and the size of the inhibition zone is measured by a vernier caliper to compare the inhibition effect of NisinA, NisinZ and nLsA proteins.
Wherein, according to GenBank ID: nisA gene sequence described in L16226.1 and GenBank ID: the nisZ gene sequence described in AF420259.1 was used to synthesize a corresponding fragment and construct an expression vector. NisinA and NisinZ were expressed and purified according to the method described in example 2. Tests are carried out according to the bacteriostasis method, and the bacteriostasis performance of the nLsA protein, NisinA and NisinZ is compared.
The bacteriostatic effect is shown in table 1, and the comparison graph of the bacteriostatic effect is shown in fig. 2.
TABLE 1
The bacteriostasis result shows that the nLsA protein has better bacteriostasis effect than NisinA and NisinZ under the same concentration.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Shandong university
<120> nLsA protein, structural gene and application thereof
<130> 202020791
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 210
<212> DNA
<213> Artificial sequence
<400> 1
atgagtacaa aagattttaa cgtttcgaag aaagatggtc catgctacat actagaacgc 60
aataaaagtt acaatattgc atcaccacgc atttcgctat gtacacccgg cattcgtggt 120
attttgaaaa gtggttgtaa aacaggagct ctgatgggtt gtaacatgaa aacagcaact 180
tgtcattgta gtattcacgt aagcaaataa 210
<210> 2
<211> 69
<212> PRT
<213> Artificial sequence
<400> 2
Met Ser Thr Lys Asp Phe Asn Val Ser Lys Lys Asp Gly Pro Cys Tyr
1 5 10 15
Ile Leu Glu Arg Asn Lys Ser Tyr Asn Ile Ala Ser Pro Arg Ile Ser
20 25 30
Leu Cys Thr Pro Gly Ile Arg Gly Ile Leu Lys Ser Gly Cys Lys Thr
35 40 45
Gly Ala Leu Met Gly Cys Asn Met Lys Thr Ala Thr Cys His Cys Ser
50 55 60
Ile His Val Ser Lys
65
<210> 3
<211> 30
<212> DNA
<213> Artificial sequence
<400> 3
tgctctagaa tgagtacaaa agattttaac 30
<210> 4
<211> 29
<212> DNA
<213> Artificial sequence
<400> 4
cccaagcttt tatttgctta cgtgaatac 29
Claims (9)
1. An nLsA protein, which is characterized in that the amino acid sequence of the nLsA protein is shown as SEQ ID No. 2.
2. A gene encoding the nLsA protein of claim 1.
3. The gene of claim 2, wherein the gene is a nucleotide sequence shown in SEQ ID No. 1.
4. A primer pair for amplifying the genes as claimed in claim 2 or 3, which is named nLsA-F and nLsA-R, wherein the nucleotide sequence of nLsA-F is shown as SEQ ID No.3, and the nucleotide sequence of nLsA-R is shown as SEQ ID No. 4.
5. A biomaterial containing the gene of claim 2, which is an expression cassette, a recombinant vector, a recombinant microorganism or a recombinant cell.
6. A method for constructing a recombinant strain containing the gene of claim 2 or 3, comprising the steps of:
vector construction: using nLsA-F and nLsA-R as primer pairs and genome of pickled vegetable bacteria as a template, amplifying to obtain nLsA gene, performing double enzyme digestion, and performing recombinant connection to obtain an nLsA expression vector;
and (3) vector transformation: transferring the nLsA expression vector into competent cells and culturing on a selective culture medium;
screening positive clones: and selecting transformants with correct sequencing, and verifying by sequencing with upstream and downstream primers of the gene, wherein the recombinant strain obtained with correct sequencing is obtained.
7. Use of the nLsA protein according to claim 1, or the gene according to claim 2 or 3, or the biomaterial according to claim 5, in the preparation of a product having bactericidal and/or bacteriostatic effects, said bacteria being micrococcus luteus.
8. A method of killing and/or inhibiting bacteria, the method comprising contacting bacteria with an effective amount of nLsA protein according to claim 1 or an article according to claim 7 for a sufficient time to be effective to kill or inhibit bacteria; the method does not include diagnosis or treatment of disease, and the bacterium is micrococcus luteus.
9. The method of claim 8, wherein the effective amount of nLsA protein is not less than 0.1 mg/mL.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EA200000758A1 (en) * | 1998-02-13 | 2001-04-23 | Пфайзер Продактс Инк. | STREPTOMYCES AVERMITILIS GENE CHANGING RELATION B2: B1 |
| CN101691397A (en) * | 2009-09-29 | 2010-04-07 | 中国科学院微生物研究所 | Nisin mutant protein, code gene thereof and application thereof |
| CN102002097A (en) * | 2009-09-03 | 2011-04-06 | 中国科学院微生物研究所 | Mutant protein capable of being combined with nisin, and coded gene and application thereof |
| CN108948160A (en) * | 2018-07-27 | 2018-12-07 | 深圳先进技术研究院 | A kind of Lactococcus lactis antibacterial peptide, preparation method and application |
-
2020
- 2020-03-06 CN CN202010151094.8A patent/CN111269296B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EA200000758A1 (en) * | 1998-02-13 | 2001-04-23 | Пфайзер Продактс Инк. | STREPTOMYCES AVERMITILIS GENE CHANGING RELATION B2: B1 |
| CN102002097A (en) * | 2009-09-03 | 2011-04-06 | 中国科学院微生物研究所 | Mutant protein capable of being combined with nisin, and coded gene and application thereof |
| CN101691397A (en) * | 2009-09-29 | 2010-04-07 | 中国科学院微生物研究所 | Nisin mutant protein, code gene thereof and application thereof |
| CN108948160A (en) * | 2018-07-27 | 2018-12-07 | 深圳先进技术研究院 | A kind of Lactococcus lactis antibacterial peptide, preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| 1株具抑菌和抗氧化活性乳酸菌的筛选及鉴定;张香美 等;《食品科学》;20170609;全文 * |
| Effects of Tylosin and Nisin on Canned Food Spoilage Bacteria;Denny C.B.et al.;《Applied and Environmental Microbiology,》;19610331;全文 * |
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